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Hunting down fungal secretomes using liquid‐phase IEF prior to high resolution 2‐DE
Author(s) -
Vincent Delphine,
Balesdent MarieHélène,
Gibon Julien,
Claverol Stéphane,
Lapaillerie Delphine,
Lomenech AnneMarie,
Blaise Françoise,
Rouxel Thierry,
Martin Francis,
Bonneu Marc,
Amselem Joëlle,
Dominguez Victoria,
Howlett Barbara J.,
Wincker Patrick,
Joets Johann,
Lebrun MarcHenri,
Plomion Christophe
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200900415
Subject(s) - proteome , mycelium , hydrophobin , leptosphaeria maculans , biology , proteomics , secretory protein , fungus , shotgun proteomics , biochemistry , microbiology and biotechnology , chromatography , chemistry , botany , secretion , gene
The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L . maculans and L . bicolor , mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1‐DE and 2‐DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2‐DE. Compared with the previously published protocols for which only dozens of 2‐D spots were recovered from fungal secretome samples, up to approximately 2000 2‐D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, β‐glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L . maculans and L . bicolor , and the first application of liquid‐phase IEF to any fungal extracts.